An accessory prefrontal cortex–thalamus circuit sculpts maternal behavior in virgin female mice

Abstract The ability to care for the young is innate and readily displayed by postpartum females after delivery to ensure offspring survival. Upon pup exposure, rodent virgin (nulliparous) females also develop parental behavior that over time becomes displayed at levels equivalent to parenting mothers. Although maternal behavior in postpartum females and the associated neurocircuits are well characterized, the neural mechanisms underlying the acquisition of maternal behavior without prior experience remain poorly understood. Here, we show that the development of maternal care behavior in response to first‐time pup exposure in virgin females is initiated by the activation of the anterior cingulate cortex (ACC). ACC activity is dependent on feedback excitation by Vglut2+/Galanin+ neurons of the centrolateral nucleus of the thalamus (CL), with their activity sufficient to display parenting behaviors. Accordingly, acute bidirectional chemogenetic manipulation of neuronal activity in the ACC facilitates or impairs the attainment of maternal behavior, exclusively in virgin females. These results reveal an ACC‐CL neurocircuit as an accessory loop in virgin females for the initiation of maternal care upon first‐time exposure to pups.


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Response to the Referees' comments on the manuscript: "An accessory prefrontal cortex-thalamus circuit sculpts maternal behavior in virgin female mice" (EMBOJ-2022-111648) Referee #1: This paper tests a preconceived notion whether the accessory prefrontal cortex is involved in a behavior of virgin female mice that resembles caregiving to newborns. They provide evidence that it does and it does via the thalamus avoiding the medial preoptic area. This latter region is a known node that is involved in pregnancy-related development for maternal behavior. The behavior that the authors aim to model in mice is a highly complex and ambiguous entity in humans, let alone in mice. The strategies they utilized are reasonable and logical and provide novel insights. Nevertheless, it is difficult to place the overall results in context of physiology, specifically that of the human condition. In addition, compared to humans, mice have a very limited development of the PFC.
We thank the Referee for the overall very positive evaluation of our manuscript, the experimental approach and the results. We are grateful to the Referee for pointing out that the need to better conceptualize our data and the overall message within the context of physiology and to specify its human relevance, if any. We have integrated these aspects into the revised manuscript in the discussion section (blue highlights). In the respective paragraph on p.12, we propose the identified ACC-CL loop to operate as an alternative route to activate the maternal care circuit, specifically the PAG for the final motor execution of this behavioural repertoire. As such, we suggest this additional circuit element to act as an enhancer to compensate for the absence of hormonal priming of the mPOA in virgin females and to enable the experience-dependent acquisition of pup retrieval behavior. In the broader context of physiology and pathobiology, our observations suggest that complementary (or even redundant) mechanisms exist to ensure engagement in offspring care via learnt strategies. From a translational perspective, our findings suggest that in conditions when mothers cannot readily take on offspring care because of a constellation of exogenous and/or endogenous influences, behavioural performance can be enhanced by recruiting non-pregnancy/non-parturition-related learning strategies.

Specific questions:
1) What is the outcome of such induction of "maternal behavior" in future natural deliveries of the same mice?
The point raised by the Referee touches on an interesting question. Our experimental design was based upon the pup retrieval test as robust and reliable readout for the evaluation of maternal behaviour. In the current settings, mothers retrieve the pups very fast. It would be unlikely that we could detect a further "improvement" in their performance, which has previously been used for the 12th Sep 2022 1st Authors' Response to Reviewers induction of maternal behaviour as virgins (floor effect). Therefore, and considering that we suggest this being a learnt behavior, our hypothesis is that once learning is consolidated, the animals will be able to retrieve their repertoire without recruiting this neurocircuit again.
2) Does it depend on the ovarian cycle how fast virgin females "acquire" this behavior?
The Referee indeed put forward a highly relevant question, which we have experimentally addressed during the revision process. Indeed, it is perceivable that the distinct hormonal levels/profiles associated with individual stages of the ovarian cycle could bias how fast virgin females acquire parental behavior. Therefore, we grouped females according to their cycle stage ("follicular stage": pre-estrus/ estrus; "secretory stage" metestrus/ diestrus) at their first pup exposure. We found no difference between the groups, suggesting that the hormonal phase indeed did not bias the performance of virgin females. These data are shown in Extended View

3) Can males be induced to develop this maternal behavior?
We thank the referee for another stimulating question, which is echoed by Referee #2. Indeed, there is evidence in the literature that male mice can develop parental behaviour, although their response to pups is highly dependent on their endogenous state (see for review: PMID 26122293). While fathers will exhibit parental care behaviour (PMID 23299896), including pup retrieval, it is well documented that virgin male mice display aggressive behaviour towards pups, most typically resulting in them attacking and finally killing the pups. This occurs because the evolutionary goal is to remove the current litter. This is achieved by stopping the female to lactate, thus accelerating the time to her next ovulation and enhancing the probability of a successful mating (PMID 7058349). Against the background of animal welfare and ethical considerations, we have decided to address your question in a setting not involving potential physical harm to the pups with their retrieval being a major outcome parameter. Instead, and also responding to a similar request by Referee #2, we have examined whether repeated contact with pups over three consecutive days (with the pups being protected from the physical attacks by the males in a wire mesh enclosure, which however allowed the male to use all classical sensory cues) would lead to an engagement of the ACC-CL circuit, comparable to the one of virgin females. We have found that the distinctive change in ACC c-Fos labelling across days in virgin females does not occur in males. Thus, we suggest sex-specific activation patterns. Nevertheless, we think these data could be over-interpreted if presented in the core manuscript. Therefore, we placed them in Extended View Fig. 4 (revised numbering).

Referee #2:
This study investigates maternal care in virgin females using pup retrieval behavior. The authors find that this behavior can be learned and that the ACC is a key mouse brain region activated during the behavior. By circuit tracing experiments, they find that the bidirectional and excitatory circuit between ACC and CL (thalamic centrolateral nucleus) is important, where Gal + neurons in the CL stimulate excitatory neurons in the ACC. The authors also use chemogenetic methods to functionally validate the ACC-CL circuit.
This study employs careful and well-controlled approaches to identify and validate the key brain regions, cell types, and circuits associated with maternal caring in virgin females.
Given that little is known about the mechanisms underlying the initial onset of maternal care behavior in virgin females, and that postpartum depression is a serious health issue, these findings are important and set a key stage for further studies.
We sincerely appreciate the favourable feedback on our manuscript and its relevance. We have considered all points raised by the Referee as detailed below.
Major comments: Figure 4 do not seem to support the specific roles of the ACC-CL(Gal) pathway. The authors could try a pathway-specific modulation or optogenetic stimulation. At the very least, the authors need to give detailed interpretations of the current results with regard to cell-type specificity.

The results from the chemogenetic experiments in
Indeed, the aspect of cell-type specificity is highly pertinent to the overall findings presented in our manuscript. We thank the Referee for motivating us to further explore this question. Therefore, we assessed the consequences of the specific chemogenetic activation of CL Gal+ neurons on pup retrieval behavior in virgins, using Gal-Cre mice (that are widely used by the community in studies on hypothalamic organization and function). Remarkably, we found that chemogenetic activation of CL Gal+ neurons in virgin females significantly shortened the latency to retrieve the pups, as compared to controls. These results corroborate those we have presented in the original manuscript (non-cell-type-specific manipulations). Data are now part of revised Figure 6, and support the specific engagement of the ACC-CL(Gal) pathway for the display of maternal behaviour in virgin females.

It is unclear why the authors did not try to modulate CL(Gal) neurons and see whether ACC neurons show altered activities and the mice show altered behavior. Such
modulations would strongly support the reciprocal and functional connectivity between ACC and CL.
As described above, the chemogenetic modulation of CL Gal+ neurons alone was indeed sufficient to bias the behaviours of virgin females.
Minor comments: 1. In Figure 1, the authors show the extent of parental behavior and ignorance ( Figure   1c,d) as percentages. The authors should clarify, in the methods section, the criteria of "ignoring" behavior, as parental and ignoring behaviors do not add up to 100% We thank the Referee for drawing our attention to this point. We have clarified the terminology and are now using the terms 'pup-directed' (instead of 'parental') and 'non-pup directed' (instead of 'ignoring') behavior in the respective description of the results on p.4 and in the corresponding methods. There, we also defined that pup-directed behaviors include crouching, licking and grooming, and covering over the pups. In contrast, non-pup directed behaviors comprise selfgrooming, eating and sleeping. Other elements related to parental care, but not involving physical interaction with the pups (eg. nest building), were not considered explaining why pup-directed and non-pup-directed behaviors do not add up to 100%. Following the Referee's suggestion, we have changed the graphical representations in Figures   4b-4d to better display the differences between the individual days.

Figures 2 and 3 panels.
As proposed by the Referee, we have added detailed labels of the brain regions shown in Figures   2 and 3.

Would the ACC-CL circuit also contribute to the acquisition of parenting behavior in male mice?
The Referee raises an interesting question, which relates to a comment brought up by Referee 1 (#3). Indeed, there is evidence in the literature that male mice can develop parental behaviour, although their response to pups is highly dependent on their endogenous state (see for review: PMID 26122293). While fathers will exhibit parental care behaviour (PMID 23299896), including pup retrieval, it is well documented that virgin male mice display aggressive behaviour towards pups, most typically resulting in them attacking and finally killing the pups. The evolutionary goal is to remove the current litter and therefore stop lactation of the female. This is expected to accelerate the time to her next ovulation, enhancing the probability of a successful mating thereafter (PMID 7058349). Against the background of animal welfare and ethical considerations, we have decided to address your question in a setting not involving potential physical harm to the pups with their retrieval being a major outcome parameter. Instead, we have examined whether repeated contact with pups over three consecutive days (with the pups being protected from the physical attacks by the males in a wire mesh enclosure, which however allowed the male to use all classical sensory cues) would lead to an engagement of the ACC-CL circuit, comparable to the one of virgin females. We have found that the distinctive change in ACC c-Fos labelling across days in virgin females does not occur in males. Thus, we suggest sex-specific activation patterns.
Nevertheless, we think these data could be over-interpreted if presented in the core manuscript.
Therefore, we placed them in Extended View Fig. 4 (revised numbering).
Overall, we thank the expert Referees their comments. We hope our answers will be accepted, particularly given the experimental follow-up we have carried out during the revision process. Thank you for submitting your revised manuscript to The EMBO Journal. Your study has now been re-reviewed by referee #2 who appreciates the introduced changes. I am therefore very pleased to let you know that we will accept the manuscript for publication here. Before sending you the formal accept letter there are a few editorial points that need to be resolved.
-we are missing 3-5 keywords -COI needs to be renamed to Disclosure and Competing Interests Statement -Please remove the Authors Contributions from the manuscript. The 'Author Contributions' section is replaced by the CRediT contributor roles taxonomy to specify the contributions of each author in the journal submission system. Please use the free text box in the 'author information' section of the manuscript submisssion system to provide more detailed descriptions (e.g., 'X provided intracellular Ca++ measurements in fig Y') -Make sure that the author checklist is completely filled out.
-Please also check panel callouts for Figure 7. Supplementary Data Table 1 is called out, but there is no Supplementary Data Table 1.
-The source data needs to be sorted as one folder per figure and ZIPed together. If you have source data for EV figures, please ZIP them together in a separate folder. See also https://www.embopress.org/pb-assets/embosite/Guide%20for%20SourceData%20Submission-1656066810500.pdf -We include a synopsis of the paper (see http://emboj.embopress.org/). Please provide me with a general summary statement and 3-5 bullet points that capture the key findings of the paper.
-We also need a summary figure for the synopsis. The size should be 550 wide by  high (pixels).
- Figure 2-D3 zoom box. Please check that the "zoom" box matches the highlighted square - Figure 4B -please check the labeling of the "zoom". I think it should be 1,2,3. Please also double check that the "zoom" box matches the highlighted box.
- Figure 6B -the highlighted box has been drawn to wide. Please redraw to reflect the zoom box.
-There are some black boxes in Figure EV5 B -Day 1 panels.
-you can only have 5 EV figures. I think EV3, EV4 & EV5 can be combined into one EV figure   -The legend for Table EV1 should also be added to the ms file -The author email bounced for Laura Cuenca Rico -laura.cuenca-rico@meduniwien.ac.at -please double check.
-Also, can you make sure to upload good resolution figures -Our publisher has also done their pre-publication check on your manuscript. When you log into the manuscript submission system you will see the file "Data Edited Manuscript file". Please look at the word file and the comments regarding the figure legends and respond to the issues.
-Please also include a point-by-point response to the editorial points when you resubmit the revised version.
That should be all -let me know if you have any further questions. And congratulations on a nice study! With best wishes Karin Karin Dumstrei, PhD Senior Editor The EMBO Journal 1 Dear Karin, please find below our replies to you editorial comments.
Thank you so much for your support, Best regards,

Daniela and Tibor
Response to the Editorial comments on the manuscript: -2022-111648R1) 1. We are missing 3-5 keywords.

"An accessory prefrontal cortex-thalamus circuit sculpts maternal behavior in virgin female mice" (EMBOJ
The key words have been added after the abstract.

COI needs to be renamed to Disclosure and Competing Interests Statement.
COI has been renamed accordingly.

Please remove the Authors Contributions from the manuscript. The 'Author
Contributions' section is replaced by the CRediT contributor roles taxonomy to specify the contributions of each author in the journal submission system. Please use the free text box in the 'author information' section of the manuscript submisssion system to provide more detailed descriptions.
The Authors contributions have been removed from the manuscript and the respective section in the journal submission system amended where necessary.

Make sure that the author checklist is completely filled out.
The author checklist has been checked and completed where required. 7. We include a synopsis of the paper (see http://emboj.embopress.org/). Please provide me with a general summary statement and 3-5 bullet points that capture the key findings of the paper.

Please also check panel callouts for Figure 7. Supplementary Data
A general summary statement and corresponding bullets points are now included in the submission.

We also need a summary figure for the synopsis. The size should be 550 wide by [200-400] high (pixels).
A summary figure with the size guidelines provided has been uploaded.

Figure 2-D3 zoom box. Please check that the "zoom" box matches the highlighted square.
The zoom box in Figure  2-D3 has been checked and corrected.
10. Figure 4B -please check the labeling of the "zoom". I think it should be 1,2,3. Please also double check that the "zoom" box matches the highlighted box.
The labelling of the zoom boxes and their matching with the highlighted box has been checked and corrected for Figure 4B.
11. Figure 6B -the highlighted box has been drawn to wide. Please redraw to reflect the zoom box. Figure 6B has been redrawn to reflect the zoom box.

The highlighted box in
12. There are some black boxes in Figure EV5 B -Day 1 panels. Figure EV5B -Day1 panels have been removed.

You can only have 5 EV figures. I think EV3, EV4 & EV5 can be combined into one EV figure.
The EV figures have been reduced to five, as EV3/ EV4 and EV5 have been combined into one EV figure as suggested.
14. The legend for Table EV1 should also be added to the ms file.
The legend for Table EV1 has been added to the ms file.

The author email bounced for Laura Cuenca Rico.
The author email for Laura Cuenca Rico has been checked and corrected.
16. Also, can you make sure to upload good resolution figures.

The resolution of the figures has been improved and good resolution figures have been uploaded.
17. Our publisher has also done their pre-publication check on your manuscript. When you log into the manuscript submission system you will see the file "Data Edited Manuscript file". Please look at the word file and the comments regarding the figure legends and respond to the issues.
We have checked the Data Edited Manuscript word file and responded to all issues raised.  Please note that it is EMBO Journal policy for the transcript of the editorial process (containing referee reports and your response letter) to be published as an online supplement to each paper. If you do NOT want this, you will need to inform the Editorial Office via email immediately. More information is available here: https://www.embopress.org/page/journal/14602075/authorguide#transparentprocess Your manuscript will be processed for publication in the journal by EMBO Press. Manuscripts in the PDF and electronic editions of The EMBO Journal will be copy edited, and you will be provided with page proofs prior to publication. Please note that supplementary information is not included in the proofs.
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